Spinning and Fully Integrated Microdevice for Rapid Screening of Vancomycin-Resistant Enterococcus.

Abstract

This study introduces a spinning and fully integrated paper-based microdevice that can perform multiple functions, including DNA extraction, amplification, and colorimetric detection, for monitoring two major vancomycin-resistant Enterococci (VREs), which carry the vanA and vanB genes. The spinning microdevice is composed of a stationary part and a spinning part. The square-shaped stationary part has two zones: the lysis and reaction zones. The spinning part, which has a spin wheel-like shape, was inserted perpendicularly into the stationary part so that its two semicircles remained on the upper and lower parts. Sodium hydroxide-treated glass microfiber filter discs, inserted in the upper semicircle, were soaked in the lysis chambers by folding them toward the lysis zone to capture DNA in the lysis chambers. The captured DNA was transferred to the reaction chambers by folding the discs toward the reaction chambers. Water was added to the sodium hydroxide-treated glass microfiber filter discs to elute purified DNA into the reaction chambers. The upper semicircle was then unfolded, and the reaction chambers were sealed for subsequent loop-mediated isothermal amplification (LAMP) for 45 min. After the reaction, the spinning part was spun in the lysis zone direction to bring the lower semicircle, inserted with phenolphthalein-treated glass microfiber filter discs, toward the upper part of the stationary part. By folding it toward the reaction chambers, the lower semicircle came into contact with them and the phenolphthalein-treated glass microfiber filter discs were soaked in the reaction chambers and expressed color after 30 s. Based on the pH change during the LAMP reaction, the phenolphthalein-treated discs remained pink in the absence of target DNA, while those in contact with the positive samples turned colorless. A sensitive detection with a VRE limit of detection of 102 CFU/mL for tap water spiked with VRE carrying the vanA gene was achieved using this microdevice. Both VREs, carrying vanA and vanB genes, were successfully identified from tap water and contaminated equipment surfaces within 75 min. The introduced microdevice demonstrated a rapid, accurate, and sensitive performance for the environmental assessment of VRE contamination in resource-limited regions.